Extensive literature exists that describes how to make large, single crystals of proteins and enzymes for research purposes such as for x-ray crystallography. The principle of most of these methods is to concentrate the enzyme very slowly during the course of several days, weeks or even months by evaporation of water from the sample. A review of these methods can be found in Journal of Crystal Growth, Volume 90 (1988), pages 1-368, which describes many of the methods and approaches considered to be of value when crystallizing biological macromolecules. None of the known methods is, however, useful for preparative or industrial scale production of large uniform crystals of enzymes.
There is a wealth of literature and knowledge on the crystallization of small molecular organic and inorganic compounds. For many small molecular compounds, it is well known how to produce large uniform crystals in evaporation or cooling batch crystallization. There are numerous methods to selectively remove small crystals from a crystallizer and thereafter collect the desired large size class of crystals. Usually the procedures include methods to maintain some degree of supersaturation until the desired size of crystals is obtained Despite the wide theoretical and practical knowledge of crystallization in general, however, the crystal growth processes that sort crystals based on size and enable production of large crystals have not been applied on the more than 1025 examples of crystallizations of biological macromolecules (G. L. Gilliland: A biological macromolecule crystallization database: a basis for a crystallization strategy. Journal of Crystal Growth vol. 90 (1988) 51-59.).
An industrial scale batch crystallization process for glucose isomerase is described in U.S. Pat. No. 4,699,882. In this method, the enzyme is crystallized in a suitable concentration of ammonium sulfate. The crystals produced are of varying size, typically 1 to 100 micrometers, and there is no control of size distribution or average size. The method is suitable for large scale production, but it is not useful for the production of large, for example, 0.5 to 1 mm crystals, or for the crystallization of such large crystals on the surface of solid inert materials.
It is therefore an object of the invention to enable an industrial scale procedure for making large enzyme crystals. Such crystals provide several advantages in that they can be used directly in columns (if they are insoluble in the substrate), and they avoid clogging or flow resistance problems that would be obtained with small crystals. In addition, enzymes produced as large crystals can be separated more easily from other materials (impurities), including solid, small particular debris such as amorphous precipitate or cell walls (that are typically 0.1-1 um in size), by screening or sedimentation and centrifugation, thus allowing their production in greater yields.
It is a further object of the invention to provide a method for depositing enzymes as crystalline layers onto surfaces of inert materials whereby the coated material can be used to catalyze specific reactions. The use of foreign materials as nuclei for crystallization provides a novel and surprisingly advantageous method of preparing immobilized enzymes. The method disclosed herein is also very useful for the growth of large enzyme crystal masses on such solid surfaces. By growing large crystals on the surface of solid materials, there is provided a simple method for enzyme recovery, immobilization and use in industrial processes.